Isotope-coded Fluorous PALs

Prof. Qisheng Zhang at UNC-Chapel Hill has published several papers about the synthesis and use of fluorous tagged photoaffinity labels (PALs) and has just published another report in Chemical Communications. The general strategy is to attach a photosensitive reactive group on a probe small molecule, incubate it with the biological sample of interest, and then expose the mixture to light. Whatever proteins or other molecules that the probe is bound to will then form an irreversible covalent bond through the photo-generated reactive group. The classic photosensitive functionality if a diazirine which upon irradiation decomposes to a reactive carbene which will usually then react with whatever bond is nearby. By using a fluorous tagged diazirine, Prof. Zhang has been able to then enrich samples for the fluorous tagged compounds through fluorous solid phase extraction (FSPE). Tagging through PALs followed by enrichment is an often used approach with the enrichment being performed primarily through affinity tags such as biotin or His-tags. Using fluorous enrichment, however, confers all of the usual advantages such as low non-selective binding, ease of elution, and excellent MS characteristics.

Besides PALs and other cross-linking strategies, another popular strategy in proteomics is isotope-coding in order to be able to directly compare different samples. For example, if you wanted to compare protein levels in healthy cells vs. diseased cells, you could add the appropriate protein labeling agent to the healthy cells and the same protein labeling agent, except with some deuteriums of 13C’s in it, to the diseased cells. Mix the two labeled samples together in equal amounts then compare the ratio of labeled proteins by MS. Most of the time, you’d see a 1:1 ratio for the proteins, but every now and then you’d see a protein overexpressed or underexpressed in the disease cell state vs the healthy state. This would then indicate that that protein may be important in the disease process.

In his group’s latest report, Prof. Zhang combines these two strategies by using a pair of isotope-encoded fluorous PALs. As seen in the figure above, the difference in the two PALs is proton vs. deuterium substitution on the aromatic ring. In practice, the authors used not the benzyl alcohol, but the NHS benzyl carbonate (not shown here) to first label a small peptide (RKRSRAE) through the side chain amine of the lysine. They took an equimolar ratio of the two in MeOH and irradiated the sample and found a near 1:1 ratio of the O-H bond insertion product of the carbene with MeOH. This demonstrated that the isotope-encoded PAL’s behaved similarly and thus suitable for their intended use.

Next the authors looked at the effectiveness of the FSPE separation. They took a 1:1 and a 2:1 mixture of the isotope-encoded insertion products from above and added them to a BSA tryptic digest. As can be seen the amount of fluorous labeled peptides before FSPE is quite small in comparison to the BSA peptides. The difference after FSPE, however, is quite dramatic and demonstrates the power of the FSPE in the sample enrichment. They also found that the original isotope ratio was largely maintained, so no preferential enrichment of the deuterated over the protonated labels.